1. Technical Field
The present invention is related to visualization of separated proteins and nucleic acids in suitable support medium. More particularly, the present invention is related to sensitive, simple and rapid visualization is biopolymer, preferably protein and nucleic acid, separated either on polyacrylamide gel or in thin membranes such as cellulose nitrate and the like.
2. Prior Art
One of the methods known for visualization of protein is described in U.S. Pat. No. 4,405,720. The stain described in this patent requires the use of three solutions and it takes a minimum of about 30 minutes to perform. Furthermore, the technique described in said patent does not stain proteins or nucleic acids in thin membranes such as cellulose nitrate.
Coomassie blue stain, the most commonly employed protein stain, takes hours to perform and it lacks the sensitivity to detect proteins present in low concentrations in biological fluids or tissues. Sensitivities achieved with heavy metal stains or fluorescent stains, on the other hand, were found to be less than, or at best, equivalent to Coomassie blue (about 10 ng of protein). Merril et al., Electrophoresis 1982, pp 327-342. Recently, more than a hundred-fold increase in sensitivity over that obtained with Coomassie staining was achieved by adapting a histological silver tissue stain for use with polyacrylamide gels. de Olmos, Brain, Behav. Evol. 2, 313-237 (1969), Switzer, et al Anal Biochem. 98, 231-237 (1979), Merril, et al Proc. Natl. Acad. Sci. USA 76 4335-4339 (1979). This stain could detect as little as a tenth of a nanogram of protein and an image could be achieved in less than 6 hours.
Histological silver stains were also adapted to visualize proteins separated by electrophoresis independently by two other groups; Kerenyi and Gallyas developed a stain for cerebrospinal fluid proteins separated on agarose Clin. Chim. Acta 38, 465-467 (1972), Clin. Chim. Acta 47 425-436 (1973) and Hubbell et al used a silver stain which was specific for nucleolar proteins Cell Biology Int'l Rep. 3, 615-622 (1979), Lischwe, et al Life Sciences 25 701-708 (1979).
The Kerenyi and Gallyas stain did not achieve widespread acceptance for a number of reasons. It was a histological stain adapted for use in agarose; it did not work well in polyacrylamide and it produced numerous staining artifacts. Merril, et al Anal. Biochem 110, 201-207 (1981), Verheecke, J. Neurol. 209, 59-63 (1975). In addition, there was a report of its quantitative irreproducibility. Verheecke, J. Neurol. 209, 59-63 (1975). Recent work by Peats has improved the Kerenyi stain performance in agarose resulting in a stain with linear protein concentration response from 0.2 ng/mm.sup.2 to 2.5 ng/mm.sup.2 and a reduction of artifacts. Peats, Biotechniques 1, 154-156 (1983).
In continuing the search for a means to simplify the methods for visualizing proteins, nucleic acids and other such entities separated on polyacrylamide gels, the Applicant reasoned that the selective reduction of silver from an ionic to a metallic form is the probable basis for both histological staining techniques and photography. Many early histological silver stains appear to have been derived from photographic methods. One of the pioneers of histological silver staining, Ranon y Cajal, credits his photographic experience with the revolutionary idea of using a photographic reducing agent (such as hydroquinone) to develop neuronal images in nervous tissue impregnated with silver nitrate. Gibson, Creative Minds in Medicine, 53-71 (1963). Recent histological silver stains have in general become more complex as empirical alterations have been made in the procedures to limit the staining to specific cells or subcellular structures. At the same time photographic image development has remained relatively simple, consisting of two steps: selective reduction of activated silver ions, followed by the removal of nonreduced silver ions. The selective reduction of silver ions may be performed by chemical reducing agents, chemical development, or by the use of light (photodevelopment).
Silver stains which have been used to visualize proteins separated on gels have utilized the "chemical development" of silver for visualization of the protein patterns. By modifying photochemical procedures a series of simple "photochemical" stains were developed. U.S. Pat. No. 4,405,720; Merril, et al, (1981) supra; Merril, et al, Science 211, 1437-1438 (1981); Merril et al, Electrophoresis 3, 17-23 (1982). These stains employed formaldehyde as the silver ion reducing agent in an alkaline solution containing sodium carbonate. The formaldehyde is oxidized to formic acid while reducing the ionic silver to metallic silver. Merril et al, (1981) supra. The formic acid produced in this process is neutralized by the sodium carbonate in the developing solution, thereby maintaining the alkaline conditions.
Silver stains utilizing photographic "chemical development" techniques have been shown to be over 100 fold more sensitive than Coomassie blue staining; they use minimal amounts of expensive reagents, and polypeptide images may be obtained within 50 minutes. Merril, et al, Anal. Biochem. 110, 201-207(1981), Science 211, 1437-1438(1981), Electrophoresis 3, 17-23(1982). Many variations of silver staining methods employing "chemical development methods" have been recently derived. These have been reviewed by Dunn and Burghes, Electrophoresis 4, 173-189(1983). It should be noted, however, that a sensitive, simple and rapid system for visualization of separated proteins and nucleic acids, both in gel electrophoresis and in thin membranes, has simply not heretofore been known in the art to which it pertains.